Experimental Biology and Medicine

Fertilization, implantation, and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-to-cell communication. This communication Involves integrins, a large family of proteins involved in the attachment, migration, invasion, and control of cellular function. Over the past decade, investigators have learned that integrins participate in multiple reproductive events including fertilization, implantation, and placentation in many species. This review will describe: (i) the expression of Integrins on gametes and during the establishment and development of the placenta; (ii) regulatory pathways for controlling expression of integrins in the uterus and developing placenta; (iii) the function of integrins as determined by null-mutations; and (iv) reproductive dysfunction in women related to inappropriate integrin expression in the uterus and/or placenta.

Insulin-like growth factor-1 (IGF-1) is an essential growth factor for normal intrauterine development and postnatal growth. Mice with a complete deficiency of IGF-1 (IGF-1-null mice), created by homologous recombination, were found to exhibit postnatal lethality, growth retardation, infertility, and profound defects in the development of major organ systems. Furthermore, IGF-1-null mice were resistant to growth hormone (GH) treatment in peri-pubertal somatic growth. Using the Cre/IoxP-induced conditional knockout system, we generated a mouse that lacks IGF-1 specifically in the liver, the primary site of IGF-1 production. Interestingly, although circulating and serum levels of IGF-1 were decreased by ≈ 75% in these mice, they exhibited no defect in growth or development. When administered exogenously, GH stimulated IGF-1 production in several extra-hepatic tissues as well as body growth. The “Somatomedin hypothesis” originally proposed that circulating IGF-1 acting in various tissues mediate the effects of GH. These striking in vivo results, obtained using homologous recombination technology, call for a major modification of the Somatomedin hypothesis. These gene targeting studies confirm that IGF-1 is essential for GH-stimulated postnatal body growth. However, liver-derived (endocrine) IGF-1 is not essential for normal postnatal growth, though it does exert a negative feedback on GH secretion. Instead, local production of IGF-1, acting in a paracrine/autocrine fashion, appears to mediate GH-induced somatic growth. This review will discuss the effects of tissue-specific IGF-1 gene deficiency created by the Cre/IoxP system versus the conventional IGF-1 knockout.

This study is a continuation of previous work searching for possible anatomic reasons to explain variable and usually unpredictable postoperative pain and dysfunction after the same nerve losses with similar neck dissection operations. The study consisted of dissections of 19 deceased unpreserved elderly subjects arterially injected with dyed latex. of the 19 subjects, 14 had brain stem and cervical spinal cord dissections, and all had neck dissections. The findings suggested two possible anatomic reasons for the pain and dysfunction: (i) The intracranial anatomy of the lower four cranial nerves, the glossopharyngeal (IX), the vagus (X), the spinal accessory (XI), and the hypoglossal (XII), was just as variable as the previously reported peripheral spinal accessory nerve plexus; and (ii) Both the intracranial and neck dissections indicated that the blood supply to the lower four cranial and cervical nerves, particularly to the brachial plexus, could be impaired by atherosclerosis and/or neuroforamlnal impingement or operative loss. This loss of blood supply theoretically could result in ischemia as another possible cause of postoperative pain and dysfunction. It is concluded that because of the potential importance of each nerve and vessel, often unknown at operation, it is very important to spare as many of them as possible to avoid subsequent painful impairment.

The baboon (Papio sp.) is an accepted nonhuman primate model for the study of the endocrinology of human pregnancy. To further characterize this model with regard to leptin function, messenger RNA transcripts for both long (Ob-RL) and short (Ob-RS) leptin receptor isoforms were Identified in maternal tissues at various stages of gestation. Thus, placental villous, subcutaneous and omental adipose tissues were collected upon cesarean delivery at early (Days 60–62), mid (Days 98–102) and late (Days 159–164) pregnancy (term ≈ 184 days). Additionally, amniochorion, decidua, and corpus luteum were collected in late gestation. Expression of Ob-RL and Ob-RS transcripts was determined in relation to constitutively expressed glyceralde-hyde-3-phosphate dehydrogenase via reverse transcriptase-polymerase chain reaction, and transcripts were localized within specific placental cell types by In situ hybridization. Ob-RL and Ob-RS transcripts were present in amniochorion, decidua, and corpus luteum at term and appeared constitutively expressed throughout gestation in placenta and adipose tissues. Ob-RS was expressed in greater (P < 0.02) abundance than Ob-RL in all tissues. Within the placenta, receptor Isoforms were localized predominantly to the syncytiotrophoblast. The expression of leptin receptor transcripts in maternal adipose tissues, as well as in the syncytiotrophoblast, amniochorion, decidua, and corpus luteum, suggests the potential for autocrine/paracrine roles for the polypeptide in the endocrinology of primate pregnancy. These are the first such observations in a nonhuman primate and support the use of the baboon as a model for the study of leptin in human pregnancy.

The prolactin (PRL) receptor (R), a member of the cytokine hemopoietin receptor superfamily, has been shown to activate early differentiation steps along the erythroid pathway. In particular PRL, a product of bone marrow stroma, induces functional erythropoietin (EPO)-R on CD34⁺ hemopoietic progenitors. In this study, expression of EPO-R mRNA and responsiveness to EPO were assessed on enriched hemopoietic progenitor cells (HPC) from seven hyperprolactinemic and three normo-prolactlnemic patients and two normal subjects. Expression of EPO-R mRNA by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was found in HPC of four out of seven hyperprolactinemic patients but not in normoprolactinemic patients or normal donors. Development of EPO-dependent Colony Forming Unlt-Erythroid (CFU-E) colonies in semi-solid medium was observed only in hyperprolactinemic patients (six out of seven). A much higher number of CFU-E colonies was observed in the four patients with a positive EPO-R message. We conclude from these data that abnormally high levels of PRL may increase the number of EPO-responsive hemopoietic precursors In vivo as they do In vitro. Since hyperprolactinemla associates in these patients with depressed EPO production, it may be regarded as a compensatory mechanism for the reduced availability of the hemopoietic factor.

Phytoestrogens are a normal constituent of soy protein and have been shown to have anti-inflammatory activity in various in vitro and in vivo models. The present study was designed to determine if a diet enriched in the phytoestrogen isoflavones, genistin and daidzin, would alter the antigen-induced cellular infiltration, particularly eosinophilia, characteristic of a guinea pig model of asthma. Throughout the duration of the study, guinea pigs were maintained on a control diet (standard guinea pig chow) or the same diet enriched in isoflavones. The animals were placed on the diet 2 weeks prior to active sensitization with ovalbumin (OA). Three weeks after sensitization, animals were challenged with OA aerosol. The cellular infiltration into the lung and protein and red blood cells (RBC) in the bronchoalveolar lavage fluid (BAL) were determined 17 hr later. In animals maintained on the control diet, OA aerosol challenge resulted in the expected increase in eosinophils in both the BAL and the lung tissue, an increase in neutrophils in the BAL, and an increase in protein and the number of RBC in the BAL. In contrast, in animals maintained on the isoflavone diet, the OA-induced eosinophilia in the lung tissue was significantly attenuated. In addition, OA challenge caused a greater increase in BAL protein in animals maintained on the isoflavone diet compared with animals on the control diet. Our results indicated that a diet enriched in isoflavones results in reduced antigen-induced eosinophilia in the lung in the guinea pig model of asthma. However, this beneficial anti-inflammatory effect of dietary phytoestrogens is accompanied by a potentially detrimental increase in antigen-induced leakage of protein into the airspace.

The ribonucleoprotein, telomerase, is responsible for the maintenance of telomere length in most Immortal and cancer cells. Telomerase appears to be a marker of human malignancy with at least 85% of human cancers expressing its activity. In the present study, we examined a series of tumor-derived and in vitro immortalized cell lines for telomerase activity levels, telomere lengths, and expression levels of the RNA and catalytic components of telomerase. We found significant variability in both telomere lengths and telomerase activity in clones from tumor cells. In addition, the levels of telomerase components or telomerase activity were not predictive of telomere length. Data from clonally derived cells suggest that critically shortened telomeres in these tumor-derived cell lines may signal activation of telomerase activity through an increase in the expression of the catalytic subunit of telomerase. Although clones with low telomerase shorten their telomeres over time, their subclones all have high levels of telomerase activity with no telomere shortening. In addition, analysis of early clones for telomerase activity Indicates substantial variability, which suggests that activity levels fluctuate in individual cells. Our data Imply that cell populations exhibit a cyclic expression of telomerase activity, which may be partially regulated by telomere shortening.

Arginine-specific mono-ADP-ribosylatlon of proteins and arginine-specific mono-ADP-ribosyltransferase occur in heart. We developed a polyclonal antiserum, R-28, against ADP-ribosyipolyarginine that recognized mono-ADP-ribosylated proteins and identified the major mono-ADP-ribosylatlon products of quail heart. Treatment of Immobilon-bound ADP-ribosylated G₂ protein with hydroxylamlne under conditions that remove ADP-ribose from Its arginines eliminated R-28 Immunoreactivity to G₂ Also, R-28 Immunoreactivity to quail heart proteins was removed by NaOH and phosphodiesterase I treatments. Similar treatment with mercuric chloride did not remove the immunoreactivity but did remove exogenously (via in vitro pertussis toxin treatment) added ADP-ribose from cysteine of cardiac G₁/G_o proteins. The antiserum did not appear to react with ADP-ribosyiasparagine of Rho (formed by C₃ toxin), ADP-ribosyidiphthamide of elongation factor 2 (formed by diphtheria toxin) in quail heart preparations, or polyADP-ribosylated proteins of a neonate rat cardiac nuclear preparation. Thus, the R-28 antiserum appears to contain predominantly antibodies directed against ADP-ribosyiarginine. To test the usefulness of R-28, immunoblotting of subcellular fractions of quail heart was performed. R-28 showed the greatest Immunoreactivity in the sarcolemma with significant Immunoreactivity in denser membrane fractions. The cytosol also contained an Immunoreactlve band distinct from those found in the membranes. Hydroxylamine treatment eliminated immunoreactivity in the sarcolemma and denser membrane fractions but not the cytosol, suggesting the membranous Immunoreactlve bands contain ADP-ribosylarginlne. In conclusion, a polyclonal antiserum that recognizes ADP-ribosyiarginine proteins has been raised. The usefulness of the antiserum is demonstrated by the characterization of endogenous arginine mono-ADP-ribosylatlon products in quail heart. The quail heart has several sarcolemmal and denser membrane fraction proteins that appear to be mono-ADP-rlbosylated on arginines.

Several epidemiological studies suggest the involvement of aluminum (AI) in the pathogenesis of Alzheimer's disease (AD). There is an increase in the levels of Aβ and ubiquitin in the pathological lesions of AD. Therefore, we have investigated whether aluminum (AI) treatment alters the levels of Aβ and ubiquitin in murine neuroblastoma (NBP2) and rat glioma (C-6) cell cultures. At a low concentration (10 μM), aluminum sulfate stimulated the level of immunoreactive Aβ and ubiquitin in NBP2 cells without changing the levels of the amyloid precursor protein (APP). However, at higher concentrations (100 and 500 μM), aluminum failed to elicit any significant effect on β-amylold, whereas ubiquitin levels continued to increase. No changes in the Aβ and ubiquitin content were found in the C-6 glioma cells following treatment with AI at any of the concentrations tested. Exposure of cells to aluminum salts did not alter the rate of proliferation in either of the two cell lines. These data suggest that one of the mechanisms by which AI may play a role in AD is by promoting the formation of Aβ and ubiquitin in neurons.